The neocortex, the largest region of the cerebral cortex, processes sensory information giving rise to perception, volitional motor responses, and to complex phenomena such as learning and memory. These distinct functions are performed by specialized 'areas'of the neocortex, characterized by unique connectivity and architecture. The continuing objective of this project is to define and study developmental mechanisms that direct the anatomical, connectional, and functional organization of the mammalian neocortex, and its parcellation into areas. The specification and differentiation of neocortical areas is likely controlled by interplay between genetic regulation intrinsic to the neocortex and extrinsic influences such as thalamocortical (TCA) input that relays sensory information from the principal sensory nuclei of dorsal thalamus. A major recent advance is evidence for the genetic regulation of arealization that implicates the transcription factors EMX2 and PAX6 by loss of function analysis of mutants for Emx2 and Pax6 (Sey/Sey). However, this role for EMX2 and PAX6 is only inferred from changes in the patterned expression of genes used as positional markers in cortex because these mutants die on P0, well before areas become anatomically and functionally defined. The role of TCA input, which has been mainly limited to the finding that embryonic patterning of marker genes is normal in the absence of TCAs, is also limited because the available mutants that lack TCAs die at P0. Thus, mechanisms regulating arealization remain poorly characterized. Our goal is to define the sufficiency and requirements for EMX2 and PAX6 as intrinsic regulators, TCA input as an extrinsic influence, We will test our hypotheses using gain of function (transgenic) and loss of function (conditional knockouts) analyses, as well as genetic cell ablations, using tissue-specific gene activation / inactivation approaches in mice designed to live to be adults. We will analyze the area-specific phenotypes of cortical neurons, including expression of markers of positional identity, area-specific input and output projections, and the anatomical and functional organization of the neocortex into areas. The multilevel analyses and alternative strategies proposed will circumvent potential problems associated with more limited approaches, provide complementary findings to substantiate interpretations and further insights into the regulation of arealization. We expect findings that support our hypotheses, but the experiments are designed such that results that disprove our hypotheses will be equally informative.